molecules Review Quinolones: Mechanism, Lethality and Their Contributions to Antibiotic Resistance Natassja G. Bush y , Isabel Diez-Santos y , Lauren R. Abbott and Anthony Maxwell * Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; [email protected] (N.G.B.); [email protected] (I.D.-S.); [email protected] (L.R.A.) * Correspondence: [email protected]; Tel.: +44-16-0345-0771 These authors contributed equally to this work. y Academic Editor: Derek J. McPhee Received: 27 October 2020; Accepted: 28 November 2020; Published: 1 December 2020 Abstract: Fluoroquinolones (FQs) are arguably among the most successful antibiotics of recent times. They have enjoyed over 30 years of clinical usage and become essential tools in the armoury of clinical treatments. FQs target the bacterial enzymes DNA gyrase and DNA topoisomerase IV, where they stabilise a covalent enzyme-DNA complex in which the DNA is cleaved in both strands. This leads to cell death and turns out to be a very effective way of killing bacteria. However, resistance to FQs is increasingly problematic, and alternative compounds are urgently needed. Here, we review the mechanisms of action of FQs and discuss the potential pathways leading to cell death. We also discuss quinolone resistance and how quinolone treatment can lead to resistance to non-quinolone antibiotics. Keywords: fluoroquinolones; DNA gyrase; topoisomerases; antibacterials; DNA topology; supercoiling; antibiotic resistance 1. Introduction The quinolone antibiotics (Figure1) are the most successful class of topoisomerase inhibitors to date. They are synthetic antimicrobials with the initial compound, nalidixic acid, being discovered as a by-product of chloroquine synthesis in 1962 [1,2]. They are used to treat bacterial infections caused by both Gram-positive and Gram-negative bacteria, including, but not limited to, urinary tract infections (UTIs), pyelonephritis, gastroenteritis, sexually-transmitted diseases, such as Gonorrhoea, tuberculosis [3], prostatitis, community-acquired pneumonia and skin and soft-tissue infections [4,5]. However, due to an increase in resistance and issues surrounding toxicity, their use in the treatment of mild infections has been contraindicated [6]. The global rise in antibiotic resistance has galvanised research into new antibiotics against both well-established targets and new targets. It has also sparked further research into antibiotics whose mode of killing is less well-established, as well as how bacteria become resistant to them. This is certainly true in the case of quinolones. In this review, the current knowledge on the mode of action of quinolones, how they kill bacteria and known pathways to resistance will be discussed. Moreover, we will review the current literature on sublethal quinolone exposure leading to resistance to quinolone and non-quinolone antibiotics. Molecules 2020, 25, 5662; doi:10.3390/molecules25235662 www.mdpi.com/journal/molecules Molecules 2020, 25, 5662 2 of 27 Molecules 2020, 25, x FOR PEER REVIEW 2 of 28 FigureFigure 1. Chemical1. Chemical structures structures of severalof several significant significant fluoroquinolones. fluoroquinolones. Nalidixic Nalidixic acid acid (the (the first first “quinolone”)“quinolone”) is shown, is shown, along along with with the chloroquinethe chloroquin by-producte by-product inspiring inspiring its synthesis. its synthesis. Note Note that that nalidixicnalidixic acid acid lacks lacks the 4-quinolonethe 4-quinolone core core and and instead instead contains contains a 1,8-naphthyridine a 1,8-naphthyridine nucleus. nucleus. 2. Background on Quinolones 2. Background on Quinolones The discovery of nalidixic acid (Figure1) was reported in 1962 during the analogue synthesis of a The discovery of nalidixic acid (Figure 1) was reported in 1962 during the analogue synthesis of lead structure: 7-chloro-1-ethyl-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid, which was detected as a lead structure: 7-chloro-1-ethyl-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid, which was detected an impurity in the synthesis of chloroquine (an antimalarial agent). From the analogues produced, as an impurity in the synthesis of chloroquine (an antimalarial agent). From the analogues produced, nalidixic acid was notable due to its moderate antibacterial activity against Gram-negative species nalidixic acid was notable due to its moderate antibacterial activity against Gram-negative species (except against Pseudomonas aeruginosa), including Escherichia coli, both in vitro and in vivo [1]. (except against Pseudomonas aeruginosa), including Escherichia coli, both in vitro and in vivo [1]. Several Several years later, nalidixic acid was released for clinical use for the treatment of uncomplicated years later, nalidixic acid was released for clinical use for the treatment of uncomplicated UTIs [7]. UTIs [7]. This sparked the synthesis of additional analogues, although with little improvement over This sparked the synthesis of additional analogues, although with little improvement over nalidixic nalidixic acid in terms of spectrum of activity and serum concentration [1,8,9]. Other analogues included acid in terms of spectrum of activity and serum concentration [1,8,9]. Other analogues included oxolinic acid, which was also introduced to the clinic, and these compounds, along with nalidixic acid oxolinic acid, which was also introduced to the clinic, and these compounds, along with nalidixic (although, in relation to its structure, nalidixic acid is a 1,8 naphthyridone and not a true quinolone [1,2]), acid (although, in relation to its structure, nalidixic acid is a 1,8 naphthyridone and not a true are considered first-generation quinolones [4,10,11]. There are many proposals concerning how the quinolone [1,2]), are considered first-generation quinolones [4,10,11]. There are many proposals generations of quinolones should be defined. Of particular note, there is the suggestion of quinolone concerning how the generations of quinolones should be defined. Of particular note, there is the generations characterised by their structure, mechanism and their killing pathway [12], and there is suggestion of quinolone generations characterised by their structure, mechanism and their killing the classification by structure, in vitro activity and clinical use [13]. In this review, we are referring pathway [12], and there is the classification by structure, in vitro activity and clinical use [13]. In this to the quinolone generations classified by their clinical uses and spectrum of antibacterial activity review, we are referring to the quinolone generations classified by their clinical uses and spectrum of outlined by Andriole [11]. Continued optimisation of the quinolones led to a fluorine atom being antibacterial activity outlined by Andriole [11]. Continued optimisation of the quinolones led to a substituted onto carbon 6 (C-6) of the quinolone scaffold (Figure3), producing a fluoroquinolone (FQ). fluorine atom being substituted onto carbon 6 (C-6) of the quinolone scaffold (Figure 2), producing a The first fluoroquinolone was Flumequine, which, after brief use in the clinic, was abandoned due fluoroquinolone (FQ). The first fluoroquinolone was Flumequine, which, after brief use in the clinic, was abandoned due to ocular toxicity [13,14]. Another key modification that enhanced potency was Molecules 2020, 25, 5662 3 of 27 to ocular toxicity [13,14]. Another key modification that enhanced potency was the incorporation of a piperazine ring onto C-7 [2,15–18]. This C-7 addition, along with the C-6 fluorine, formed the second-generation quinolones, which have a broader scope of activity and better bioavailability, as well as improved pharmacokinetic and pharmacodynamic properties [8,16,19–21]. They were also less toxic and were less susceptible to single point mutations that led to high levels of resistance seen against the first-generation quinolones [7,8,17,22]. The second-generation quinolone class began with norfloxacin (Figure1)[ 15], which proved to be effective in the treatment of genitourinary and gastrointestinal tract infections, as well as increased activity against P. aeruginosa [15,16,21–23]. However, it was ciprofloxacin (Figure1) that was the first quinolone that showed e ffective systemic activity [8,17,24,25]. Ciprofloxacin is listed as a first-line treatment for low-risk febrile neutropenia within cancer patients and a second-line treatment for cholera, as well as being employed clinically against a range of UTIs, such as those caused by Pseudomonas aeruginosa [26]. It has also been demonstrated to be effective in the treatment of Enterobacteriaceae-induced osteomyelitis, prostatitis and septicaemia [8]. Following the success of ciprofloxacin, the observed structure-activity relationships (SARs) were explored further (Figure3). This medicinal chemistry e ffort produced a wide range of newer-generation FQs (third and fourth generations) that have even broader spectra of activity, greater efficacy and a lower prevalence of resistance [27]. Sparfloxacin and moxifloxacin (Figure1) are the better-known compounds of the third and fourth generations, respectively, and are amongst the first quinolones to show significant potency against Gram-positive bacteria [11]. Furthermore, Mycobacterium tuberculosis, the bacterial species that causes tuberculosis (TB; currently the world’s deadliest bacterial infectious disease to date), is susceptible to the FQs, and both moxifloxacin and levofloxacin (a second-generation quinolone)